Chymotrypsin tertiary structure

Blow, D.M., Chymotrypsin tertiary structure and enzymic activity. Biochem J 1968, 110, 2P. 

All peptidases within a family will have a similar tertiary structure, and it is not uncommon for peptidases in one family to have a similar structure to peptidases in another family, even though there is no significant sequence similarity. Families of peptidases with similar structures and the same order of active site residues are included in the same clan. A clan name consists of two letters, the first representing the catalytic type as before, but with the extra letter P , and the second assigned sequentially. Unlike families, a clan may contain peptidases of more than one catalytic type. So far this has only been seen for peptidases with protein nucleophiles, and these clans are named with an initial P . Only three such clans are known. Clan PA includes peptidases with a chymotrypsin-like fold, which besides serine peptidases such as chymotrypsin... 

This method was employed for transesterification reactions with both a-chymotrypsin and subtilisin Carlsberg with a variety of H+/Na+ buffers [53]. With both enzymes (which differ widely in secondary and tertiary structures) and two polar solvents, acetonitrile and THF, the activating effect of the solid-state buffer was clearly evident (Table 3.3). The observation that a variety of buffer pairs show success in activating two dissimilar enzymes in synthetically useful solvents makes this method for activation promising and novel. 

The mammalian serine proteases have a common tertiary structure as well as a common function. The enzymes are so called because they have a uniquely reactive serine residue that reacts irreversibly with organophosphates such as diisopropyl fluorophosphate. The major pancreatic enzymes—trypsin, chymotrypsin, and elastase—are kinetically very similar, catalyzing the hydrolysis of peptides... 

Fig. 5. Protein folding. The unfolded polypeptide chain collapses and assembles to form simple structural motifs such as p-sheets and a-helices by nucleation-condensation mechanisms involving the formation of hydrogen bonds and van der Waal s interactions. Small proteins (eg, chymotrypsin inhibitor 2) attain their final (tertiary) structure in this way. Larger proteins and multiple protein assemblies aggregate by recognition and docking of multiple domains (eg, p-barrels, a-helix bundles), often displaying positive cooperativity. Many noncovalent interactions, including hydrogen bonding, van der Waal s and electrostatic interactions, and the hydrophobic effect are exploited to create the final, compact protein assembly. Further structural...

Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, at a concentration as low as 0.003 M, and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed... 

Trichophyton), S34 (HflA endopeptidase), S38 (Treponema chymotrypsin-like endo-peptidase), S39 (cocksfoot mottle virus endopeptidase), S43 (porin) cannot yet be assigned to clans, since neither the tertiary structure nor the order of catalytic residues are known. 

Jornvall and Harris (91) presented data for the structures around all of the 14 cysteine residues in each protein chain. Analysis by Jornvall (92,93) of different peptide mixtures obtained after treatment of the protein with trypsin (before or after maleylation), chymotrypsin, pepsin, cyanogen bromide, or thermolysin yielded amino acid sequence information for all parts of the subunit and the primary structure of the whole protein chain was deduced (5S). It was found to contain 374 residues and is shown in Table I. An acetylated serine residue is at the N-terminus and the reactive cysteine residue is at position 46. Some residues are unevenly distributed (PS). Six of the seven histidine residues are in the N-terminal half of the molecule, the two tryptophan residues are in either terminal region, the four tyrosine residues are in the middle of the primary structure, and none of the 14 cysteine residues occur in the C-terminal quarter of the molecule. A characteristic distribution of hydrophobic residues was also noticed (93), which may now be partly correlated with the presence of large hydrophobic cores in the tertiary structure of the protein (Section II,C,3). Most regions of the primary structure were analyzed in many different overlapping peptides (92-9 ) with a corresponding increase in reliability. The structure is in excellent agreement with the total composition determined by acid hydrolysis (93). It is compatible with independently determined partial structures of... 

The changes in primary structure that accompany the conversion of chymo-tiypsinogen to a-chymotrypsin bring about changes in the tertiary structure. The enzyme is active because of its tertiary structure, just as the zymogen is inactive because of its tertiary structure. The three-dimensional structure of... 

The sulfur atom binds readily to heavy meted ions. Under oxidizing conditions, two cysteines can join together in a disulfide bond to form the amino acid cystine. When cystines are part of a protein, insulin for example, this stabiUzes tertiary structure and makes the protein more resistant to denaturation disulfide bridges are therefore common in proteins that have to function in harsh environments including digestive enzymes (e.g., pepsin and chymotrypsin) and structural proteins (e.g., keratin). Disulfides are also found in peptides too small to hold a stable shape on their own (e.g., insulin). 

Further examples of conformational adaptationf will be given, each one quite individual in its mechanism, yet collectively supplying a picture of considerable uniformity. Ribonuclease forms a compact structure, hydrophilic outside, lipophilic inside, with a slot to receive the substrate. The active site makes use of histidine residues (numbers 12 and 119) that would otherwise be remote from one another (Kartha, Bello and Marker, 1967). The dimensions of the folded ribonuclease molecule are about 30X 30X 38 A (mol. wt. 15 000). When charged with a substrate, e.g. cytidine phosphate, one histidine residue binds the phosphate group, the other the sugar. The tertiary structure of a-chymotrypsin has been similarly worked out the active site depends on the closeness of two amino acids that are on different strands, namely serine-195 and histidine-57... 

Subtilisin, a bacterial proteolytic enzyme originally isolated from Bacillt4S subtilus, is a serine protease. Even though its primary and tertiary structures bear no discernible relationship to chymotrypsin, the active site groups and the... 

The primary and tertiary structures of E. are very similar to those of the other pancreas proteinases. Of the 240 amino acids in E. (Af, 25,700), 52 % are identical to those in trypsin and chymotrypsin A and B. These include the catalytically important residues HiS57, Aspio2 and Seri, , the ion pair Vali Aspj, which is important for the conformation, and the 4 disulfide bridges. As might be expected, the 3-dimensional structure of E. is very similar to that of the other pancreatic serine proteases (see Chymotrypsin, Trypsin). 

The proteinases chymotrypsin and trypsin are two enzymes for which secondary and tertiary structures have been elucidated by x-ray analysis and which have structures supporting the lock and key hypothesis to a certain extent. The binding site in chymotrypsin and trypsin is a three-dimensional hydrophobic pocket (Fig. 2.11). Bulky amino acid residues such as aromatic amino acids fit neatly into the pocket (chymotrypsin. Fig. 2.11a), as do substrates with lysyl or arginyl residues (trypsin. Fig. 2.11b). Instead of Ser, the trypsin peptide chain has Asp which is present in the deep cleft in the form of a carboxylate anion and which attracts the positively charged lysyl or arginyl residues of the substrate. Thus, the substrate is stabilized and realigned by its peptide bond to face the enzyme s Ser which participates in hydrolysis (transforming locus). 

Also important is the finding that not only the conformer is inert to hydrolysis by a-chymotrypsin, but it also failed to inhibit enzymatic hydrolysis of the active S,Seq conformer. In marked contrast, l-KCTI has been shown to strongly inhibit chymotryptic hydrolysis of d-KCTI. This pattern of competitive inhibition has also been demonstrated for other enantiomeric pairs of chymotrypsin substrates. To understand this behavior it should be realized that the two conformers of Belleau s compound differ in two important aspects orientation of the carbomethoxyl group and the chirality of the biphenyl system. Consequently, it must be concluded that in its reaction with this constrained substrate, a-chymotrypsin displays specific recognition of molecular asymmetry. This is referred to as tertiary structural specificity. The specificity of the biphenyl compound thus serves to extend the concept that appropriately constrained substrates can serve as very useful tools. 

The enzyme is small, having a polypeptide chain of 129 amino acids. It was the first one, in 1967, to have its tertiary structure elucidated by X-ray crystallography (108). Unlike a-chymotrypsin, lysozyme has a well-defined deep cleft running down one side of the ellipsoidal molecule for binding the substrate. 

---